Apparatus with probe

ABSTRACT

An apparatus adapted to obtain a profile of a density gradient sample independently of fractionation is provided. The apparatus includes a light source, a probe comprising a first probe needle actuatable to extend into a tube containing a sample, a first light-transmitting means to receive light from the light source and transmit light through the sample as the probe needle extends into the sample, a second light-transmitting means to receive light transmitted by the first light-transmitting means and transmit the received light to a signal-producing means capable of translating the received light into a recordable signal to produce a profile of the sample. The apparatus may additionally be adapted to fractionate the sample following generation of the gradient profile.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 11/984,764, filed on Nov. 21, 2007, and thisapplication also claims the benefit of U.S. Provisional Application No.60/860,217, filed on Nov. 21, 2006, the entire contents of which priorapplications are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to an apparatus useful to probe a solutiongradient. In particular, the present invention relates to an apparatuscomprising a probe adapted to generate a gradient profile of a densitygradient independent of fractionation.

BACKGROUND OF THE INVENTION

Solution gradients or density gradients are utilized in biochemicalresearch to separate macromolecules such as proteins, DNA and RNA, andlarger aggregates such as viruses and cells. More recently, densitygradient centrifugation has found application in the field ofnanotechnology. Researchers at Northwestern University have usedgradients to separate and purify different classes of carbon nanotubes.

Solution gradients usually utilize a solute of varying concentrations toaid in the separation of particles. Examples of appropriate solutes are:sucrose, glycerol, CsCl, Optiprep™, Percoll™, ficoll, metrizamide,Nycodenz.™ and/or sodium acetate. Particles are separated duringcentrifugation either by their velocity of sedimentation, or by theirdensity if there is an isopycnic point within the solution column in thetube. Faster, or denser particles, respectively, will appear lower inthe tube.

After the sample has been subjected into the appropriate densitygradient in the centrifuge, the particles are recovered from thegradient for analysis. Fractionation methods and apparatus used torecover the sample in the gradient involve the transfer of the entiregradient or certain layers or bands of the solution gradient to othervessels. It is often desired to extract only desired bands from thesolution gradient for electron microscopy, liquid scintillation or gelelectrophoresis.

One of the earliest and simplest methods of fractionation is to piercethe bottom of the centrifuge tube with a fine bevelled needle andcollect the drops of the solution gradient as it flows through theneedle into a second vessel. The flow of the solution into the openingof the needle becomes conical. In other words, the particles directly infront of the needle opening and within a zone best described as aninverted cone above the needle are drawn into the needle opening beforeparticles outside the cone. The resulting fractionation of differentlayers of the solution gradient significantly degrades the resolutionachieved in the gradient.

Bottom puncture with side hole needles have also been used forfractionation. Side hole needles have a hole on each side of the needletip. Side hole needles are more effective than the bevelled needle, butside hole needles also draw the solution into the needle in a conicalfashion preventing high resolution of the fractionation.

One of the most common methods for fractionating solution gradientsintroduces a dense solution at the bottom of the centrifuge tube, whichfloats the gradient up to an inverted collection funnel placed on thetop of the gradient. Some loss of resolution results from theretardation of particles near the tube wall during this upward movement,and at any but the slowest flow rates, the shallow collection cone failsto prevent the shallow collection cone fails to prevent the samecone-shaped extraction of liquid directly below the cone's centralorifice experienced by the bevelled needle described above. The resultis mixing of different layers in the gradient and the resultant loss ofresolution.

These problems were addressed in U.S. Pat. No. 4,003,834 to Coombs,issued Jan. 18, 1977, an apparatus is disclosed for the fractionation ofa solution gradient by displacement with a piston, and in U.S. Pat. No.5,645,715 to Coombs issued 1995 which discloses a piston collection tipwith a unique trumpet shape collection face. The use of a piston todisplace the gradient from the top down solves the problem of particlesadhering to the wall during the upward movement of the entire gradientsince the gradient remains stationary until it is displaced by thedownward movement of the piston. The trumpet tip prevents thecone-shaped mixing by gradually compressing horizontal bands into thinvertical columns prior to collection. Tubing carrying both air and rinseis disposed within the piston to allow for cleaning of the collectiontubing, further improving resolution by preventing cross contaminationbetween fractions. Pumping air into the piston tip transfers anysolution gradient left in the tubing to a second vessel.

U.S. Pat. No. 4,003,834 also provides a means for visualizing bands ofparticles large enough to scatter visible light. However, many particlesof interest are too small to scatter visible light or are present at toolow a concentration to be detected. Since the nucleic acids and proteinsfound in these particles absorb UV light in the 260-280 nm range, it isthe current practice to detect bands of these particles by passing thegradient outflow through a UV flow cell as is frequently done in HPLCand FPLC. The UV gradient profile obtained by the flow cell can be usedas a diagnostic tool in its own right; however, in this application, theprofile is generated as the gradient is being removed from thecentrifuge tube.

There are two potential problems with this type of UV-basedfractionation. Firstly, it is difficult to accurately and reproduciblyidentify the beginning and end of UV absorbance peaks (bands) in theprofile as it is being generated. Secondly, unless the user manuallyinterrupts the flow at the start and end of each peak, the fractioncollector typically used to separate the gradient outflow into discretefractions is doing so at a constant time interval or rate of flow. Thus,there is no relationship between the peaks of absorbance and thefractions and this requires the user to scan a range of fractions toidentify those containing the particles of interest. Some UV-basedcollection systems have “peak-picking” algorithms built into theirsoftware so that rapid changes in UV absorbance in the outflow triggersample collection into a new vessel. While providing adequate separationof discrete peaks of particles, these devices have difficulty detectingand separating overlapping peaks or shoulders. Volume- or time-basedfractionation of the UV-flow cell output is disrupted by peak-picking,so the overall sampling profile is then lost. Thus, one must choosebetween obtaining uniform size samples for analysis or isolating peaks,as they are mutually exclusive.

Certain inventions (i.e. U.S. Pat. Nos. 4,873,875; 6,479,239) haveattempted to obtain a UV or fluorescent profile of the contents of acentrifuged gradient by vertically scanning the gradient with a beam oflight from the outside of the tube. These have not seen widespread usebecause the only centrifuge tubes that can withstand the severe stressof ultracentrifugation (100,000-1,000,000×g) are made of a UV-absorbingplastic, effectively preventing the beam from penetrating the tube.Consequently, the only devices currently capable of producing a UVprofile of a gradient are those which pass the gradient through adetached UV flow cell.

It would be desirable, thus, to develop a means of generating a gradientprofile independent of fractionation that may be used as a guide tofractionation.

SUMMARY OF THE INVENTION

An apparatus has now been developed which is useful to obtain a profileof a density gradient. The apparatus comprises a probe adapted to obtaina gradient profile optically. The probe advantageously causes minimaldisturbance of the gradient and thereby provides a gradient profile ofhigh resolution.

Thus, in one embodiment, an apparatus is provided adapted to obtain agradient profile. The apparatus includes a light source, a probecomprising a first probe needle actuatable to extend into a tubecontaining the gradient, said probe being in communication with thelight source and comprising a first light-transmitting means to receivelight from the light source and transmit light through the gradient asthe probe needle extends into the gradient, and a secondlight-transmitting means to receive light transmitted by said firstlight-transmitting means and transmit the received light to asignal-producing means.

In another aspect of the present invention, there is provided afractionation apparatus adapted to obtain a gradient profile of adensity gradient independently of fractionation. The fractionationapparatus comprises:

a light source;

a probe comprising a first probe needle actuatable to extend into a tubecontaining the gradient, said probe being in communication with thelight source and comprising a first light-transmitting means to receivelight from the light source and transmit light through the gradient asthe probe needle extends into the gradient, and a secondlight-transmitting means to receive light transmitted by said firstlight-transmitting means and transmit the received light;

a signal-producing means positioned to receive light from the secondlight-transmitting means, wherein said signal-producing means translatesthe received light into a recordable signal to produce a profile of thegradient; and

a piston actuatable to extend into the gradient-containing tube and tofractionate the gradient according to the sample profile;

wherein the probe and piston are moveable between a resting positionwherein the probe and piston are clear of the gradient tube, a probingposition wherein the probe is in a gradient access position and afractionating position wherein the piston is in a gradient accessposition, said probe and piston being independently actuatable.

In another aspect of the present invention, a method of fractionating agradient is provided comprising:

i) determining the profile of a gradient;

ii) selecting a fractionation plan according to the profile; and

iii) executing fractionation of the gradient according to the plan.

In a further aspect of the invention an apparatus adapted to obtain aprofile of a stationary density gradient sample using fluorescence isprovided. The apparatus comprises:

a light source;

a probe comprising a first probe needle actuatable to extend into a tubecontaining the gradient, said probe being in communication with thelight source and comprising a first light-transmitting means to receivelight from the light source and transmit light having an excitationwavelength into the gradient as the probe needle extends into thegradient, and a second light-transmitting means to transmit receivedlight to a signal-producing means, wherein the first light transmittingmeans comprises a transmitting fibre optic bundle and a first reflectionmeans and the second light-transmitting means comprises a secondreflection means and a receiving fibre optic bundle, said light beingtransmitted by the transmitting fibre optic bundle and reflected by thefirst reflection means into the gradient at an angle of about 90° to thefirst probe needle, wherein the light strikes particles in the gradientand causes the particles to emit light at an emission wavelength whichis received by said second light-transmitting means and is reflected bysaid second reflection means to said receiving fibre optic bundle andtransmitted by said receiving fibre optic bundle to a signal-producingmeans; and

a signal-producing means adapted to receive light from the secondlight-transmitting means, said signal-producing means comprising aphotodetector to translate the light into a recordable signal to producea profile of the gradient.

These and other aspects of the present invention will become apparent byreference to the detailed description, and the drawings in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an apparatus according to an aspect ofthe invention;

FIG. 2 is a side view of the fractionation portion of an apparatus ofFIG. 1 and an exploded view (A) of the valves therein;

FIG. 3 is a perspective view of the probe portion of an apparatus ofFIG. 1 and exploded views of the tip of the probe needle (A) and lightsource/photodetector (B);

FIG. 4 is a top view illustrating the arc of movement of a probe, pistonand actuator of an apparatus of FIG. 1;

FIG. 5 illustrates the first probing position (A/B) and secondfractionating position (C/D) of the apparatus of FIG. 1;

FIG. 6 illustrates an embodiment of the invention;

FIG. 7 illustrates an embodiment of the invention;

FIG. 8 illustrates an embodiment of the invention;

FIG. 9 illustrates an embodiment of the invention

FIG. 10 illustrates different geometries of the probe needle tip;

FIG. 11 illustrates the output signal including zone marking of anapparatus of FIG. 1; and

FIG. 12 illustrates the probe (B) and piston (C) of an embodiment of theinvention extended within a sample tube.

DETAILED DESCRIPTION OF THE INVENTION

A fractionation apparatus 100 is provided as shown in FIG. 1 comprisinga fractionation or collection portion 110 and a probe portion 120 usefulto generate a profile of a density gradient that can be converted into afractionation run. The fractionation portion 110 of the apparatus 100generally corresponds with that described in U.S. Pat. No. 5,645,715,the relevant portion of which (found in columns 3-7) is incorporatedherein by reference. As described, and as generally shown in FIG. 2, thefractionation portion 110 comprises a piston 10 having an internalpassageway or valve 12, and inserted within the valve 12 are acollection tube 14, an air tube 16 and a rinse tube 18. Mounted belowthe valve 12 on the end of the piston 10 is a collection tip 19, whichmay optionally be an interchangeable collection tip as described in U.S.Pat. No. 5,645,715.

As one of skill in the art will appreciate, the configuration describedin U.S. Pat. No. 5,645,715 may be modified as shown in FIG. 2 (A) toincrease efficiency. For example, the valve 12 may incorporate twoone-way valves to permit the rinsing and drying of the sample tubingwithout disturbing the gradient. The first one-way valve is a ball valve3 which prevents backflow of air or rinse into the gradient, while thesecond one-way valve is a rubber duckbill 4 valve which preventsbackflow of rinse and gradient into the air tubing. Backflow of air andgradient into the rinse line is prevented by a rubber duckbill one-wayvalve mounted in the tubing between the rinse pump and the piston.

Referring to FIGS. 1 and 3, the fractionation apparatus 100 of anembodiment of the invention comprises a probe portion 120 situatedadjacent to the piston 10. The probe portion 120 comprises a probe 20.The probe 20 consists of at least one hollow probe needle 22 which isopen at both ends. The probe needle 22 has an upper end 24, which issecured to a mounting block 27, and a lower end 26. The probe 20 andpiston 10 are both mounted on a platform or swing arm 17. The swing arm17 is mounted onto an actuator 15. As shown in FIG. 4, the center of thepiston 10 and the center of the probe needle(s) 22 are positioned on thesame radial arc 21. The arc is centered on the actuator 15 such thatrotation of the actuator 15 positions either the probe 20 or the piston10 over the gradient tube holder 50 (FIG. 1) of the apparatus 100. Thus,as shown in FIG. 5, the swing arm 17 and actuator 15 are moveablebetween a fully retracted resting position in which both the piston 10and probe 20 are clear of a tube holder 50 which holds the gradient, afirst or probe position in which the probe 20 is in position centeredabove tube holder 50 (FIG. 5(A)) and a second or fractionating positionin which the piston 10 is positioned centered above the tube holder 50(FIG. 5(C)).

The actuator 15 also functions to lower each of the probe 20 and piston10, respectively, when in position above the tube holder 50, into asample gradient tube 13 as illustrated in FIG. 9. The actuator 15 may bemanual, as illustrated and described in U.S. Pat. No. 4,003,834, therelevant disclosure of which (e.g. columns 3-7) is incorporated hereinby reference. Alternatively, the apparatus may be fully automated byincorporating a computer to drive a stepper motor to rotate an acmescrew which raises and lowers the actuator 15, providing means forprecisely determining the position and velocity of both the piston 10and the probe 20. This ensures that the extension of the probe 20 intothe sample is conducted at a constant velocity such that each data pointin the UV profile is coupled with its precise position in the gradient.

The probe needle 22 contains fiber optic bundles 28. In one embodiment,as illustrated in FIG. 6, the fibre optic bundles 28 within the probeneedle 22 include a mixed and randomized fibre optic transmitting bundle29 and a fibre optic receiving bundle 30 (FIG. 6B), each of which extendthe length of the probe needle 22. The transmitting bundle 29 isconnected to a suitable light source 32, such as an LED emitting in aselected wavelength range. For detection of UV-absorbing particles, thetransmitting bundle 29 connects to a suitable UV source such as anultra-violet light emitting diode (UV LED). In alternative embodiments,the light source may be an LED emitting in the visible range. The lightsource 32 is mounted onto the mounting block 27. A photodetector 42 isalso mounted on the mounted block 27 approximately adjacent to the lightsource 32 (FIG. 6A).

As one of skill in the art will appreciate, each fibre optic bundle willincorporate fibres manufactured of material appropriate for thetransmission of the wavelength of the light emitted from the lightsource 32. For example, if the light source 32 emits in the UV rangefrom 250 to 350 nm, quartz (fused silica) fibres may be used. The numberand diameter of the fibres in the fibre optic bundle is optimizedempirically to provide the highest signal to noise ratio and the highestresolution in a given application. For example, in certain embodiments,such as those illustrated in FIGS. 6-9, 80 fibres with 0.1 mm diameterare utilized. In these embodiments, the choice of 80 fibres was based onthe fact that fewer fibres produce a weaker signal while more fibresrequire a larger diameter needle and result in greater disturbanceduring the probing of the gradient. In certain embodiments, such asthose illustrated in FIGS. 6, 8 and 9, the fibres are split into twoindependent bundles, a transmitting bundle 29 and a receiving bundle 30.

A reflection means 34 is permanently attached to the bottom end 26 ofthe probe needle 22 which functions to reflect a beam of light receivedfrom the light source 32, conducted the length of the probe needle 22 bythe transmitting bundle 29, into the gradient. The reflecting means 34reflects the light beam into the gradient towards a second reflectionmeans 36, for example, at a 90° angle to the probe needle 22. Thereflected beam exits the needle 22, travels a gap 38 through thegradient and is then deflected by the second reflection means 36 backalong the same plane towards the first reflection means 34, e.g. at anangle of 180°. The gap 38 between the first and second reflection means34, 36 is sufficient to render a suitably accurate reading of thegradient. The first and second reflection means 34, 36 may be any meanscapable of reflecting the light beam at the required angle. In thisembodiment, for example, a prism is appropriate for use as the firstreflection means 34 having a 45° reflecting angle. While many sizes ofprism will be suitable, the prism exemplified in one embodiment hascross section dimensions of: 1.0 mm.×1.0 mm. on both 90° faces and 1.4mm along the hypotenuse reflector surface. The length of the prismmatches the length of the end of the needle, e.g. 2.9 mm.

The second reflection means 36 is affixed to a support 45 sufficient toposition it appropriately from the probe needle 22. The support 45 maybe a support needle (as shown in FIG. 3) generally aligned parallel tothe probe needle 22 such that it is appropriately spaced from the needle22 to provide gap 38. Generally, the gap 38 between the first and secondreflection means 34, 36 is in the range of 1-10 mm. Since an increase ingap size will result in increased absorbance, and a decrease in gap sizewill result in a stronger signal, the gap 38 between the first andsecond reflection means 34, 36 may be adjusted in order to maximizeresolution in view of the variability among gradients. The supportneedle 45 containing the reflecting means 36 is shown mounted ontomounting block 27 such that it will co-extend into the gradientsimultaneously with the probe needle 22 on actuation of actuator 15.

The second reflecting means 36 comprises a 180° reflecting angle and maybe, for example, a planar mirror. Its minimum dimensions are thedimensions of the beam it is to reflect, for example, 0.3×2.6 mm, butmay, of course be larger to reduce the stringency of its positioning onthe support needle 45. As indicated above, the second reflection means36 is positioned to reflect the incident beam from the probe needle 22back and into the receiving fibres 30 within the end of the probe needle22.

As shown in FIG. 10, the geometry of the probe needle end 26 has asignificant impact on the resolving power of the probe needle 22. In oneexample, the transmitting/receiving fibre bundle 29, 30 may be circular48 (FIG. 10A) at the bottom end 26 of the probe needle 22, producing acylindrical beam of light 49 that is reflected by the first reflectingmeans 34 across the gap 38 to the second reflecting means 36. The bandof particles in the gradient 51 will first be detected when the bottomedge of the cylindrical beam 49 first encounters the band 51. The bandwill be detected until the top edge of the beam 52 leaves the band,giving a total distance of detection 53. If the probe needle 22 isflattened at its lower end 26 to a rectangular shape 54 (FIG. 10B), thelight beam 55 crossing the gap 38 is much thinner, resulting in asmaller total distance of detection 56 and increased resolution. Thedimensions of the rectangle are constrained by the number, diameter andarrangement of the fibers in the bundle. For example, using a bundle of80 fibers generates a beam having 0.3×2.9 mm rectangular shape. If asingle row of fibers is used in a flattened needle 57 (FIG. 10C), thethinnest beam of light 58 is produced and (theoretically) the smallesttotal distance of detection 59. However, the amount of light availablefor detection is also much reduced (30% of the two other versions shownsince the number of fibres is decreased), so the signal to noise ratiosuffers. If the number of fibres remains the same as in the circular andrectangular examples (FIG. 10A/B) (48 and 54), the end of the needleshown in FIG. 10C would be 8 mm across, giving a greater wetted surfacearea and producing more disturbance of the gradient during insertion andwithdrawal of the probe. Thus, the dimensions of the end of the probeneedle impact on both resolution and gradient disturbance, and requireselection in order to provide an appropriate balance. Dimensions of 0.3mm×2.9 mm represent an example of a suitable compromise betweenresolution and disturbance. While bands of particles in a gradient areoccasionally 1 mm thick, most bands lie in the 2-5 mm range ofthickness, so the 0.3 mm thickness produces a tolerable loss ofresolution.

The required electronic circuitry 60 to send and receive the lightsignal is attached directly to the mounting block 27 (as shown in FIG.3A) to minimize the sensitivity of the photoreceptor to spuriouselectronic interference and vibration.

The light beam received and conducted by the receiving fibres 30 istransmitted for detection by a photodetector 42 as shown in FIG. 3. Onesuitable photodetector for UV light is a 1 mm² SiC chip contained in asmall can with a quartz window (JEC 1S, Boston Electronics, Boston,Mass.). Since the end of the receiving bundle 30 cannot be physicallycoupled to this chip, and since the light beam diverges at an angle of,for e.g., 17° after it leaves the upper end of the receiving bundle 30the light beam is transmitted onto a set of condensor lenses 40 whichcollect and refocus the beam onto the SiC chip inside the can. Anexample of a suitable set of lenses consists of two plano convex lenses(01LQF005, f=10.0 mm, dia=5 mm, Melles Griot, Ontario, Canada) arrangedas shown in the exploded view FIG. 3B.

The photodetector 42 translates the light beam into a recordable outputsuch as current or voltage which is then digitized by a microprocessor61 such as a Burr Brown microprocessor (DDC-112) and displayed on adisplay unit 44, such as a monitor, which is connected to the controlpanel 43 (FIG. 1). The absorbance values collected at regularuser-determined intervals, for example, 10 data points/mm, are stored asa spreadsheet associated with the depth in the gradient from which theyare taken. The display unit 44 functions in real-time to display thegradient UV absorbance profile throughout the depth of the gradient.

Using the real-time profile, the fractionation is planned by dividingthe profile into fractionation zones and by further dividing each zoneinto a desired number of fractions. This can be accomplished using, forexample, a rotary encoder with push switch 63 to move a vertical linecursor across the displayed profile, pressing it down to set theposition of the various zones. To synchronize the probing andfractionation functions, both stages of analysis begin with the actuator15 in its full up resting position where it contacts a limit switch. Foreach different size tube, for example Beckman's SW28, SW28.1, SW40,SW41, SW55, SW60 and TLS55, the precise vertical offset between the tipof the probe and the point of gradient capture inside the piston tip isdetermined empirically and stored in memory. Thus, the position of thecursor on the display can be precisely translated into a correspondingpiston position during fractionation. Likewise, the position of eachzone and the sub-fractions within each zone can readily be calculated.Once the plan has been set on the display, the computer converts thefractionation plan into a series of downward movements of the pistoninterrupted by a user-selected rinse protocol at the end of eachfraction. The speed of the piston's movements is set automatically, butis user-adjustable, with 0.3 mm/sec being a typical speed.

In practice, a gradient profile is obtained using an apparatus accordingto an aspect of the invention by placing a gradient-containing tube 13into the tube holder 50. As shown in FIG. 5, the probe 20 is swung intoposition above the tube and actuated to probe the sample. The probe andsupport needles (22, 45) are simultaneously lowered into the gradient ata constant speed within the range of 0.1-6.0 mm/sec. As the needlesextend into the gradient, light transmitted from the light source 32 iscaptured by the transmitting fibre optic bundle 29 and is conducted thelength of the probe needle 22 to the bottom end 26 thereof where it isreflected at a 90° angle off of the first reflecting means 34. Thereflected light travels the gap 38 through the gradient and is reflectedat a 180° angle off of the second reflecting means 36. The reflectedlight re-travels the gap 38, is reflected 90° by the first reflectingmeans 34, is captured by the receiving fibre optic bundle 30 and isconducted the length of the probe needle 22 to the top end 24 thereof.The returning beam leaves the end of the receiving bundle and iscaptured by a set of condensor lenses 40 which refocus it for detectionby the photodetector 42. The photodetector 42 translates the lightreceived into recordable output that may be displayed graphically,numerically or otherwise on a display unit 44. Upon reaching the bottomof the sample tube, the needles are withdrawn automatically.

The planning stage involves using a combined rotary encoder+push switch63 connected to the display unit 44, for example, as is a standardfeature of any oscilloscope, to move a vertical line cursor along theX-axis of the displayed gradient profile to set the various zones forfractionation. As the user sets each new zone along the profile, theuser is prompted to set the number of fractions within that zone asillustrated in FIG. 11. For example, the gradient may be a single zonedivided into a number of equal fractions. Alternatively, the gradientmay be divided into several zones with a variable number of fractionsper zone. This latter mode permits the isolation of individual bands ofparticles in the gradient for further analysis.

Once the fractionation zones and fractions are selected, the piston 10is swung into position over the gradient tube and the gradient is thenfractionated automatically. The fractionation plan developed by the userin the previous stage is converted by the computer into a series ofdownward movements of the actuator 15 and piston 10, interrupted by theinsertion of brief bursts of rinses and air to expel sample left in thetubing and remove any cross contamination between fractions.

This profiling function of the probe 20 of the present apparatus 100advantageously provides a means to view the sample gradient andparticles of interest therein, and thus, a means to plan thefractionation of the gradient prior to the actual fractionation.

To this point, an embodiment is described in which the apparatus 100comprises a dual needle probe (22, 45) with one needle (22) containingboth the sending and receiving light bundles (29, 30) and the othersupport needle (45) having mounted thereon a second reflecting means(36) to deflect the beam reflected by the first reflecting means (34)from the transmitting bundle (29) back to the receiving bundle (30),thus effectively doubling the path length of the probe.

In another embodiment of the present invention, as shown in FIG. 7, theprobe 20 includes dual probe needles, a first transmitting probe needle22 containing a transmitting fibre optic bundle 29 and a secondreceiving probe needle 23 containing a receiving fibre optic bundle 30.Both bundles will contain an appropriate number of fibers to permit amaximum beam strength and sensitivity, for example, 80 fibres each. Asset out above, the first and second probe needles are spaced by anadjustable gap 38 as shown in FIG. 7B. In this case, the firstreflecting means 34 is attached to the bottom of the first probe needle22 such that the light beam passing through the first probe needle 22 isreflected at a 90° angle through the gradient across gap 38. Thereflected light beam is received by the second reflecting means 36 whichis attached to the bottom of the receiving probe needle 23 such that itreflects the incoming light beam at an angle of 90° to be received bythe receiving fibre optic bundle 30 in the receiving probe needle 23. Asdescribed, the receiving fibre optic bundle 30 carries the light beam tothe top end of the second probe needle 23 where it is transmitted onto aset of lenses 40 for refocusing and then translation by a photodetector42 to an output signal that forms the profile of the gradient (FIG. 7A).The fractionation plan and fractionation are then executed as described.

FIG. 8 illustrates another embodiment of the present invention, in whichthe apparatus 100 incorporates a dual wavelength probe. In thisembodiment, the first transmitting probe needle 22 contains two fiberbundles of 40 fibers each. A first wavelength fibre bundle 29 a is incommunication with a first LED light source 32 a that generates light ata first wavelength and a second wavelength fibre bundle 29 b is incommunication with a second LED light source 32 b that generates lightat a second wavelength (FIG. 8A). For example, in the case of UV light,the first light source may generate UV light at a wavelength of 260 nmfor detection of nucleic acids, while the second light source maygenerate UV light at a wavelength of 280 nm for detection of proteins.The second receiving probe needle 23 contains a single receiving fibreoptic bundle 30 of 80 fibers. In this embodiment, the first and secondlight sources 32 a, 32 b transmit light in alternating pulses, which areabsorbed by the appropriate transmitting bundle, reflected by the firstreflecting means 34 to the second reflecting means 36, reflected by thesecond reflecting means 36, received and transmitted by the receivingfibre optic bundle 30 in the second receiving probe needle 23. The lightreceived by the photodetector 42 at the top of the receiving needle 23is sorted by wavelength into two data streams by the microprocessor 61(e.g. Burr-Brown processor DDC-112), one for each wavelength, generatinga dual wavelength profile at the output stage. The fractionation planand fractionation are then executed as described.

A further embodiment of the present invention is illustrated in FIG. 9.This embodiment comprises a single needle, dual bundle probe; however,rather than measuring the amount of light absorbed by particles in thegradient, this probe is designed to detect particles within the gradientor sample by fluorescence. In this embodiment, the single probe needle22 houses both transmitting and receiving fibre optic bundles 29, 30consisting of, for example, 40 fibers each (FIG. 9A). The light source32 transmits an excitation beam which is carried by the transmittingfibre optic bundle 29 and reflected into the gradient by the firstreflecting means 34, e.g. a prism with an appropriate reflecting angle(such as 45°), attached at the bottom of the probe needle 22 aspreviously described. Particles of interest 39 within the solution, suchas microbes or proteins, are detected when they absorb light from theexcitation beam having a desired excitation wavelength and then emitlight at a different wavelength, e.g. an emission wavelength. The lightemitted by the particles 39 strikes the reflecting means 34, isreflected therefrom for receipt by the receiving fibre optic bundle 30and transmitted to the photodetector 42 for translation as an outputsignal (FIG. 9B). A filtering means, such as a narrow bandpass filter,47, placed between the two condensor lenses 40 in front of thephotodetector 42 will block light of the excitation wavelength fromreaching the photodetector 42, allowing only light of the emissionwavelength to pass, as is standard practice in any commercialfluorometer. This configuration can detect particles by their naturalfluorescence, by the enhanced fluorescence of a wide variety ofcommercial dyes that bind specifically to biological molecules ofinterest or by fluorescent dye-tagged antibodies. For example, virusescan be detected using a DNA-binding dye called PicoGreen (MolecularProbes, Invitrogen, USA) (for example, see “Quantitation of AdenovirusDNA and Virus Particles with the PicoGreen Fluorescent Dye, Murakami P.;McCaman M. T. Analytical Biochemistry, Volume 274, Number 2, October1999, pp. 283-288”). The excitation light source for PicoGreen deliveredby the transmitting bundle 29 is at 485 nm and the emission wavelengthreceived by the receiving bundle 30 in the same probe needle 22 is 538or 518 nm. These dyes offer sensitivity that is reportedly 10,000 timesmore sensitive than UV absorbance. Using these dyes converts thisabsorbance device into a fluorometer probe. A new series of fluorescentdyes (CF™ dyes from Biotium, USA, <www.biotium.com>) have been coupledto antibodies for ultrasensitive detection of target proteins in largermacromolecular assemblies and cells.

As one of skill in the art will appreciate, the embodiment of FIG. 9 mayalternatively comprise separate needles (similar to the embodiment shownin FIG. 7), a first needle 22 housing the transmitting bundle 29 and asecond needle 23 housing the receiving bundle 30. In this embodiment,the light source 32 transmits an excitation beam which is carried by thetransmitting fibre optic bundle 29 and reflected into the gradient bythe first reflecting means 34 attached at the bottom of the probe needle22 as previously described. Particles of interest 39 within the solutionabsorb light from the excitation beam having an excitation wavelengthand then emit light at a different wavelength, e.g. an emissionwavelength. The light emitted by the particles 39 strikes the secondreflecting means 36, e.g. a prism with an appropriate reflecting angle(such as 45°), attached at the bottom of the second needle 23, isreflected therefrom for receipt by the receiving fibre optic bundle 30and transmitted to the photodetector 42 for translation as an outputsignal.

As will be appreciated by one of skill in the art, the present apparatus100 may be modified to solely incorporate a probe portion 120 without afractionation portion 110 as partially shown in FIG. 12B. Such a probingapparatus comprises the features detailed in the foregoing for theprobing function of the apparatus and lacks those features necessary toconduct fractionation. Thus, in instances where the desired end productof a gradient-based analyses is the absorbance profile, an apparatusincorporating only a probe portion as described herein may be used.

Although the disclosure describes and illustrates the preferredembodiments of the invention, it is understood that the invention is notlimited to these particular embodiments. Many variations andmodifications will occur to those skilled in the art. For definition ofthe invention, reference is made to the appended claims.

References referred to herein are incorporated by reference.

1. An apparatus adapted to obtain a profile of a stationary densitygradient sample, the apparatus comprising: a fluorescent light source; aprobe comprising a first probe needle actuatable to extend into a tubecontaining the gradient, said probe being in communication with thelight source and comprising a first light-transmitting means to receivelight from the light source and transmit light having an excitationwavelength into the gradient as the probe needle extends into thegradient, and a second light-transmitting means to transmit receivedlight to a signal-producing means, wherein the first light transmittingmeans comprises a transmitting fibre optic bundle and a first reflectionmeans and the second light-transmitting means comprises a secondreflection means and a receiving fibre optic bundle, said light beingtransmitted by the transmitting fibre optic bundle and reflected by thefirst reflection means into the gradient at an angle of about 90° to thefirst probe needle, wherein the light strikes particles in the gradientand causes the particles to emit light at an emission wavelength whichis received by said second light-transmitting means and is reflected bysaid second reflection means to said receiving fibre optic bundle andtransmitted by said receiving fibre optic bundle to a signal-producingmeans; and a signal-producing means adapted to receive light from thesecond light-transmitting means, said signal-producing means comprisinga photodetector to translate the light into a recordable signal toproduce a profile of the gradient.
 2. The apparatus as defined in claim1, wherein the first and second light transmitting means are housed inthe first probe needle.
 3. The apparatus as defined in claim 2, whereinthe first reflection means is also the second reflection means.
 4. Theapparatus as defined in claim 1, wherein the second light transmittingmeans is housed in a second needle that is simultaneously actuatablewith the first needle.
 5. The apparatus as defined in claim 1, whereinthe first and second reflection means is a prism with a 45 degreereflecting angle.
 6. The apparatus as defined in claim 4, wherein thefirst and second reflection means is a prism with a 45 degree reflectingangle.
 7. The apparatus as defined in claim 1, additionally comprisingmeans to prevent light of the excitation wavelength from receipt by thephotodetector.
 8. The apparatus as defined in claim 7, wherein saidfilter means is a bandpass filter.